Multimeric proteins comprising two or more structured cysteine loop-defined domains have therapeutic use in their ability to bind to a target molecule, for example, to initiate a cell signaling cascade, to block the interaction of an cognate receptor-ligand pair, or to target a pharmacological moiety. The design and use of numerous examples of such multimeric polypeptides have been described, including multimeric polypeptides that specifically bind to interleukins (e.g., IL-6), intracellular signaling cascade proteins (e.g., c-MET kinase), cell surface receptors (e.g., CD40) and cell surface adhesion molecules (i.e., integrins) (e.g., VLA-4), and cell surface co-stimulatory molecules (e.g., ICOS). See, for example, U.S. Patent Publication Nos.: 2003/0082630; 2003/0157561; 2005/0048512; 2005/0053973; 2005/0089932; 2005/0164301; 2006/0008844 and co-pending U.S. patent application Ser. Nos. 11/281,245 and 11/281,256, the disclosures of each of which are hereby incorporated herein by reference in their entirety for all purposes.
An important characteristic of the discrete monomer domains of these multimeric proteins includes their ability to fold independently of the other domains in the same protein. Folding of these domains may require limited assistance from, e.g., a chaperonin(s) (e.g., a receptor-associated protein (RAP)), a metal ion(s), or a co-factor. The ability to fold independently prevents misfolding of the domain when it is inserted into a new protein or a new environment, and contributes to heat stability of the individual domains as well as the full-length protein.
Proteins that contain these domains are involved in a variety of processes, such as cellular transporters, cholesterol movement, signal transduction and signaling functions which are involved in development and neurotransmission. See, Herz, (2001) Trends in Neurosciences 24(4):193-195; Goldstein and Brown, (2001) Science 292: 1310-1312. The function of a discrete monomer domain is often specific but it also contributes to the overall activity of the protein or polypeptide. For example, the LDL-receptor class A domain (also referred to as a class A module, a complement type repeat or an A-domain) is involved in ligand binding while the gamma-carboxyglumatic acid (Gla) domain which is found in the vitamin-K-dependent blood coagulation proteins is involved in high-affinity binding to phospholipid membranes. Other discrete monomer domains include, e.g., the epidermal growth factor (EGF)-like domain in tissue-type plasminogen activator which mediates binding to liver cells and thereby regulates the clearance of this fibrinolytic enzyme from the circulation and the cytoplasmic tail of the LDL-receptor which is involved in receptor-mediated endocytosis.
It is advantageous to develop methods for efficiently and cost-effectively producing multimeric proteins having two or more cysteine-defined loop domains, especially processes that are amenable to large-scale production. The present invention addresses this and other needs.